1.) If possible, perfuse tissue in situ with 1X PBS followed by 2%
paraformaldehyde made in PBS (directions available at CBI). Place
tissue in 2% paraformaldehyde for and additional 1-2 hours. The tissue
needs to be completely submerged in the solution. Larger tissues need 2
hours incubation in 2% paraformaldehyde in PBS (lobe of liver). One
hour is sufficient for a small tissue such as mouse lymph node or
hollow organs like rodent intestines.

2.) Place tissue in 30% sucrose for 24 hours having changed the sucrose
2-3x within 24 hours. Provide enough 30% sucrose to submerge the
tissue.†

3.) Place a plastic beaker containing 2-Methylbutane (Fisher Cat No.
O3551-4) in an ice bucket, Styrofoam box or equivalent container
containing a few inches of liquid nitrogen. Allow the beaker to cool
for several minutes. Beaker with 2-methylbutane is to remain in the ice
bucket submerged in the liquid nitrogen during the entire freezing
protocol.

4.) Samples, fixed as above or fresh, can be placed on small pieces of
filter paper manipulated with forceps (the tissue often freezes to the
forceps if picked up directly). Alternatively, the tissue sample can be
embedded in OCT compound in a small cassette.

5.) Pick up sample and completely immerse in the liquid nitrogen cooled
2-Methylbutane for 30 seconds.

6.) Remove sample from beaker and immerse in the liquid nitrogen for an
additional 10 seconds.

7.) Immerse a properly labeled, suitable box or cryopreservation bag in
the liquid nitrogen for a few seconds to cool container to the same
temperature as the tissue.

8.) Place sample into the storage container/bag and immerse in the
liquid nitrogen until placing sample into -80oC storage.

9.) Samples to be transferred between labs or waiting sectioning should
be stored on dry ice to avoid thawing.